Bottom Line:
The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization.Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization.Together, our results suggest that U2 internal modification occurs within the nucleolus.

ABSTRACTU2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5' trimethylated guanosine cap, 13 pseudouridines, and 10 2'-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.

Figure 7: (A) Structures of Xenopus wild-type U7 snRNA and MIDU2-U7 chimera are shown schematically. The U7 Sm binding site is indicated by a hatched box. Thin lines represent the U7 RNA chain. In the MID U2-U7 chimera, the first 18 nucleotides of U7 are substituted with a Xenopus U2 sequence (nucleotides 19–46) indicated by a thicker line. (B) The U7 Sm binding site also supports U2 internal modification. [α32P]UTP uniformly labeled wild-type U7 (lane 1), U2-U7 chimera (MID U2-U7; lane 2), or wild-type U2 (lane 3) was injected into isolated nuclei under oil. 5 h later, RNAs were recovered and assayed for modification. The positions of uridylate and pseudouridylate are indicated. (C) Intranuclear localization of U7 and the U2-U7 chimera. 32P- and fluorescently labeled wild-type U7 and MID U2-U7 chimera were synthesized by in vitro transcription and 1 fmol of each RNA was microinjected into the nuclei of Xenopus oocytes. Nuclei were isolated 5 h later and nuclear spreads were prepared. Spreads were also prepared from uninjected oocytes as controls. DIC and fluorescence (FL) images are shown for each field. The arrowheads in the DIC panels point to Cajal bodies. Prominent Cajal body labeling was observed for both U7 and the MID U2-U7 chimera. Weak but above background nucleolar signal was also detected for the MID U2-U7 chimera. (D) The nucleocytoplasmic distribution of U7 and the U2-U7 chimera RNAs was determined as described in the legend to Fig. 4 B. Bar, 10 μm.

Mentions:
Although our observations suggest that the nucleolus is the site of U2 internal modification, U2 constructs that become efficiently modified are detected in both nucleoli and Cajal bodies. We attempted to assess any potential role of Cajal bodies in modification by fusing a 5′ fragment (nucleotides 19–46) of U2 RNA to a 3′ half sequence of U7 snRNA, which contains a distinct Sm binding site known to target U7 snRNAs to Cajal bodies (Wu et al. 1996). Surprisingly, this U2-U7 chimera became modified (Fig. 7 A, lane 3). As expected, the RNA localized to Cajal bodies (Fig. 7 B, MIDU2-U7). However, we also detected nucleolar signals that were weak but distinctly greater than background, suggesting that these U2-U7 chimeric constructs may transiently cycle through nucleoli for modification (see Discussion).

Figure 7: (A) Structures of Xenopus wild-type U7 snRNA and MIDU2-U7 chimera are shown schematically. The U7 Sm binding site is indicated by a hatched box. Thin lines represent the U7 RNA chain. In the MID U2-U7 chimera, the first 18 nucleotides of U7 are substituted with a Xenopus U2 sequence (nucleotides 19–46) indicated by a thicker line. (B) The U7 Sm binding site also supports U2 internal modification. [α32P]UTP uniformly labeled wild-type U7 (lane 1), U2-U7 chimera (MID U2-U7; lane 2), or wild-type U2 (lane 3) was injected into isolated nuclei under oil. 5 h later, RNAs were recovered and assayed for modification. The positions of uridylate and pseudouridylate are indicated. (C) Intranuclear localization of U7 and the U2-U7 chimera. 32P- and fluorescently labeled wild-type U7 and MID U2-U7 chimera were synthesized by in vitro transcription and 1 fmol of each RNA was microinjected into the nuclei of Xenopus oocytes. Nuclei were isolated 5 h later and nuclear spreads were prepared. Spreads were also prepared from uninjected oocytes as controls. DIC and fluorescence (FL) images are shown for each field. The arrowheads in the DIC panels point to Cajal bodies. Prominent Cajal body labeling was observed for both U7 and the MID U2-U7 chimera. Weak but above background nucleolar signal was also detected for the MID U2-U7 chimera. (D) The nucleocytoplasmic distribution of U7 and the U2-U7 chimera RNAs was determined as described in the legend to Fig. 4 B. Bar, 10 μm.

Mentions:
Although our observations suggest that the nucleolus is the site of U2 internal modification, U2 constructs that become efficiently modified are detected in both nucleoli and Cajal bodies. We attempted to assess any potential role of Cajal bodies in modification by fusing a 5′ fragment (nucleotides 19–46) of U2 RNA to a 3′ half sequence of U7 snRNA, which contains a distinct Sm binding site known to target U7 snRNAs to Cajal bodies (Wu et al. 1996). Surprisingly, this U2-U7 chimera became modified (Fig. 7 A, lane 3). As expected, the RNA localized to Cajal bodies (Fig. 7 B, MIDU2-U7). However, we also detected nucleolar signals that were weak but distinctly greater than background, suggesting that these U2-U7 chimeric constructs may transiently cycle through nucleoli for modification (see Discussion).

Bottom Line:
The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization.Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization.Together, our results suggest that U2 internal modification occurs within the nucleolus.

ABSTRACTU2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5' trimethylated guanosine cap, 13 pseudouridines, and 10 2'-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.